The proposed research has two primary objectives: an understanding of the mechanism of action of thrombin as a hydrolase, and an understanding of how thrombin interacts with the vascular system, particularly the microcirculation. We will continue studies of a genetically abnormal thrombin, Thrombin Quick, which has a catalytic defect seen primarily in the acylation step, and a binding defect observed only with the large physiologic substrate, fibrinogen. We will use model amide substrates, prothrombin, antithrombin III, and fibrinogen fragments to help determine why binding to some substrates is normal, and to others is not. The magnitude of the acylation defect will be studied in the pre-steady state with model chromogenic substrates. Reactivity of active site residues will be measured with known active-site directed alkylators and acylators. Interaction of thrombin with the vessel wall (endothelium) will be studied both in vitro and in vivo. We will study (A) the mechanism by which binding of thrombin to endothelium in the microcirculation influences thrombin clearance, (B) The nature of the site that interacts with thrombin and antithrombin III. (C) The biochemistry of a second thrombin binding site that regulates the activation of the vitamin K-dependent anticoagulant, Protein C. (D) We will study the mechanism by which thrombin induces release of arachidonate, and consequently prostacyclin, from endothelium. We will ask how thrombin acts at the cell membrane to elicit the response, and what is the pathway to the release of arachidonate. We also will ask if the physiology of the interactions of thrombin in the microcirculation is important for arachidonate release.